Structural insights into the mechanism of phosphate recognition and transport by human XPR1

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Human XPR1 variants lead to cerebral calcium-phosphate deposition, which are associated with a neurodegenerative disorder known as primary familial brain calcification (PFBC) 5 . Here, we present the Cryo-EM structure of human XPR1 bound to Pi ions. XPR1 contains 10 transmembrane α-helices, forming an ion channel-like architecture that recognizes and transports Pi ions. Two arginine residues, subject to pathogenic mutation in PFBC families, line the translocation channel and serve to bind Pi ions. Clinically linked mutations of these arginine residues impair the Pi transport activity of XPR1. To track the movement of Pi ions within the translocation channel, we capture a mutant XPR1 in an alternative conformation. It reveals a rearrangement of intrahelical hydrogen bonds between a channel-lining tryptophan and two Pi-binding residues. This rearrangement links Pi recognition and transport, by means of flipping the tryptophan residue to propel Pi through the translocation channel. Our results provide mechanistic understanding of how XPR1 recognizes and transports phosphate ions across cell membrane, and they establish a framework for interpreting disease-related mutations and for the development of future therapeutics. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Biochemistry/Proteins/Membrane proteins Figures Figure 1 Figure 2 Figure 3 Introduction Inorganic phosphate (Pi) is essential for life, used wildly in biomolecules synthesis, cell metabolism and energy supply. To nourish cell, humans uptake Pi by the solute carrier 20 (SLC20) and SLC34 transporter families 6-10 . In parallel, cell exports intracellular Pi to regulate Pi homeostasis and prevent the cytotoxicity caused by Pi accumulation. Elevated Pi levels cause major disorders, with severe biochemical and clinical consequences 11-13 . XPR1 is the only known exporter that transports Pi out of cell in humans 1 , expressed in all tissues 14 . Loss-of-function variants of XPR1 accumulate cytosolic Pi and lead to cerebral calcium-phosphate deposition, which are associated with a genetic disease known as primary familial brain calcification (PFBC) 5 , 15-17 . PFBC is an adult-onset neurodegenerative disorder characterized by a large clinically heterogeneous symptoms including dystonia, parkinsonism, dementia, depression, chorea and others, yet no targeting drugs and specific treatment are currently available 18-20 . XPR1 is abundant in human platelets, where inhibiting its transport activity increases the risk of thrombosis in mouse models 21 . XPR1 deficiency in ovarian cancer cell causes toxic accumulation of cytosolic Pi, leading to cell death 22 . Dysregulation of Pi efflux could thus presents a new, potential strategy for anticancer therapy 22 , 23 . Given the significance and pathomechanisms of XPR1, it presents a promising therapeutic target for diseases and cancers 5 , 22 , 24 , 25 . However, the molecular basis by which XPR1 recognizes and transports Pi across cell membrane remains unknown. We therefore set out to determine the Cryo-EM structure of human XPR1 bound to Pi. Results The overall Cryo-EM structure XPR1 contains a transmembrane domain and a cytoplasmic N-terminal SPX domain. The transmembrane domain is responsible for facilitating Pi efflux, while the SPX domain enhances this function by binding inositol pyrophosphate (PP-InsP) stimuli 1 , 26-29 . By preparing XPR1 in the presence of PP-InsP (Supplementary Fig. 1, and Methods), we could collect high-quality cryo-EM images for single particle analysis, which allowed us to reconstruct a density map with an overall resolution of 3.3 Å (Supplementary Fig. 2a). The EM density of SPX domain is poor (Supplementary Fig. 3a), probably owing to its mobility. In contrast, the density map of transmembrane domain is clear (Supplementary Fig. 3a). Although the transmembrane domain is small, with a molecule weight of only approximately 43 kDa, the well resolved EM density enables us to build the atomic model (Supplementary Fig. 3b, c, and Supplementary Table 1). The transmembrane domain of XPR1 contains 10 transmembrane α-helices (TM1-10) and folds into two structurally distinct sub-domains, with N and C termini on the intracellular side (Fig. 1). We refer to the N-terminal portion as N domain, that is formed by TM1-TM5 and a short amphipathic helix (AH) lying parallel to the membrane. The C-terminal portion harbors the conserved EXS (named for homologous regions found in yeast ERD1 and SYG1 and human XPR1) domain 30 , 31 (Supplementary Fig. 4), that is made up of TM6-TM10. The EXS domain associates with TM5, creating a pore that spans across the membrane (Fig. 1a). Lined the membrane-spanning pore, two phosphate ions were identified (Fig. 1a, Supplementary Fig. 3d), implying that the pore serves to transport Pi. Notably, TM9b positions close to the central pore axis on the extracellular side (Fig. 1a), suggesting its potential role in Pi exporting. A 3-dimensional structural homology search with the program DALI 32 found no known structures similar to the XPR1 transmembrane domain, indicating a specific mechanism for phosphate recognition and transport in XPR1. The structure basis for phosphate recognition and transport HOLE 33 analysis for the solvent-accessible pathway of XPR1 transmembrane domain reveals that it exhibits an ion channel-like architecture for Pi transit (Fig. 2a, b). The intracellular entrance of the translocation channel is formed by TM5a, TM6-TM8 and TM10 (Fig. 2a). Owing to a kink in TM9, the channel is bent and created by TM9b, TM10, TM6, TM5b and the tip of TM2 on the extracellular side. The narrowest point of the channel has a radius of approximately 1.2 Å, which exceeds the water access limit (1 Å) 34 , indicating that the channel is solvent-permeable. The entrance and the interior wall of the first half of the channel are positively charged, while the second half and the exit are negatively charged (Fig. 2b). Given the anionic property of Pi ions, the presence of polarized electrostatic potential across the translocation channel may serve to move Pi ions in and out. The solvent-accessibility of the translocation channel (Fig. 2a), together with aligned Pi ions along the permeation pathway (Fig. 2b), suggests that XPR1 may export Pi ions through this channel without requiring large conformational changes as observed in Pi importers. Pi importers exhibit an outward-open conformation for Pi uptake from extracellular side and undergo large conformational changes to adopt an inward-open conformation, allowing for Pi release into cytosol 35-37 . This conformational transition is driven by the energy derived from movements of other co-translocated ions (e.g., Na + or protons) down their concentration gradients 35-37 . In contrast, the Pi export activity of XPR1 is independent of pH gradient across the cell membrane 3 , 27 , and no co-translocated ions have been found 23 . These previously observed phenomena are now supported and aligned with by the channel-like architecture of XPR1. Our structure provides a framework for understanding the mechanism by which XPR1 recognizes phosphate. Two phosphate ions are recognized by a series of polar residues in the translocation channel (Fig. 2c). One of them (Pi 1 ) interacts with the side chains of N401, R570, Q576 and E600, and is coordinated downstream (intracellular-to-extracellular direction) of the other Pi ion (Pi 2 ). The Pi 2 is recognized by interacting with D398, K482, Y483, D533, R570, R603, R604 and W607. Additionally, the side chain of R459 positions the orientation of D398 and Y483, facilitating them to coordinate Pi 2 in the binding site. Moreover, the side chain of R570 points towards the midpoint between the two Pi ions, bridging them, which implies its potential role in Pi delivery during translocation. There is a discontinuity in TM9 that is kinked at W573. The TM9b is stabilized by an intrahelical hydrogen bond between W573 and Q576, as well as the interaction between Q576 and Pi 1 . These Pi-recognizing residues are conserved in different species (Supplementary Fig. 4). To assess the roles of the above-mentioned key residues in Pi transport, we reconstituted XPR1 into liposomes and performed Pi transport assays (Methods). By substituted the Pi 2 -recognizing residues D398, K482, D533, R603 and R604 with alanine, and Y483 and W607 with phenylalanine, and could purify the R603A or the Y483F in sufficient quantities to perform the proteoliposome-based transport assays. We find that XPR1 carrying either R603A or Y483F substitution have impaired Pi transport activities (Fig. 2d), suggesting crucial roles of Y483 and R603 in transporting Pi. Moreover, consistent with the structure configuration of the Pi 1 binding site (Fig. 2c), replacing side chain of N401, Q576 or E600 with alanine resulted in a reduced Pi transport activity of XPR1 (Fig. 2d). Clinically linked residues in XPR1 function in phosphate recognition and transport XPR1 is a causative gene identified in primary familial brain calcification (PFBC) families 5 , 16 , 17 , and the R459C and R570C missense variants have been found to be pathogenic 15 , 20 , 38 , 39 . Our structure reveals that R459 is involved in maintaining the binding network of Pi 2 , and R570 forms salt bridges with Pi 2 and Pi 1 (Fig. 2c). Substituting the side chains of R459 and R570 might perturb Pi binding and potentially impair XPR1 activity. Indeed, we find that the PFBC families variants, R570C and R459C, exhibit an significant reduction in Pi transport activity (Fig. 2d). Therefore, our structure and function analysis provide mechanistic insights into the correlation between patient mutations and XPR1 function. The mechanism for linking phosphate recognition and transport How do the phosphate ions pass through the XPR1 transmembrane channel? A structure comparison between channel alone and bound to phosphate ions would provide mechanistic insights. With the resolved experimental structure of the phosphate-bound channel at hand, we compared it with the unbound state model predicted by AlphaFold 40 . While the AlphaFold model and our experimental structure share the overall fold, a notable disparity is observed in the TM9 segment (Supplementary Fig. 5). The experimental structure has a kink in this segment, causing a shift of TM9b towards the central membrane-spanning pore axis, while the AlphaFold model predicts an unbent TM9. We reasoned that the TM9 kink in the experimental structure is stabilized by the intrahelical hydrogen bond formed between the side chains of Q576 and W573, and the interaction between Q576 and the bound phosphate (Pi 1 ) (Fig. 2c). We therefore speculated that substituting Q576 side chain with alanine would disrupt the intrahelical hydrogen bond and lead to the Pi 1 dissociation. To clarify these envisioned conformational changes, we sought to determine the Cryo-EM structure of the Q576A mutant XPR1. The transmembrane domain structure of the XPR1 Q576A mutant was resolved at a resolution of 3.2 Å (Supplementary Fig. 2b, and Methods). This mutant presents a similar overall structure to that of wild-type, and also two phosphate ions are observed in the translocation channel (Supplementary Fig. 6). Notably, local conformational changes in the Pi 1 binding site are revealed (Fig. 3a). In the mutant structure, the Pi 1 ion undergoes a forward movement (intracellular-to-extracellular direction) within the translocation channel. Consequently, the hydrogen bonds between Pi 1 and the side chains of N401 and E600 are broken. Moreover, the indole ring of W573 flips to form an intrahelical hydrogen bond with R570. This rearranged intrahelical hydrogen bond disrupts the salt bridge between R570 and Pi 1 , and thus the Pi 1 dissociation and forward movement. Additionally, given the high density of π-electrons in the W573 indole ring, the ring-flipping and closing to the Pi 1 ion would push the anion forward. The ring-flipping therefore links Pi recognition to transport. Supporting this, when the indole ring of W573 is substituted with polar or hydrophobic group like W573N, W573Y, W573A and W573L, the Pi transport activity of XPR1 is reduced (Fig. 3b). Together, the translocation channel likely employ a "ring flipping-push-movement" mechanism to propel Pi ions through, facilitated by the rearrangement of two intrahelical hydrogen bonds between W573-Q576 and W573-R570 (Fig. 3c). As Q576 is replaced by alanine in the mutant structure, a possibility cannot be ruled out that the side chain of Q576 also plays role in the Pi forward movement in native protein. Discussion Elevated levels of cytosolic phosphate are cytotoxic due to its presence as a potent metal chelator and a pervasive inhibitor of cellular enzymes. XPR1 and its evolutionarily conserved orthologues are the proteins that transport cytosolic phosphate out of cells 1 – 3 , 47 , 48 . A very recent study discovered that the XPR1 orthologue in fruit fly (Pxo) lowers cellular phosphate levels, by generating a newly defined phosphate-storing organelle (PXo body) and transporting cytosolic phosphate into the PXo body 4 . These transporters belong to the structurally uncharacterized SLC53 family. Here the experimental structure of XPR1 transmembrane domain, together with its multiple functional states bound to phosphate, provides the first snapshots for understanding the structure-function relationships of SLC53 family transporters. XPR1 mutations are linked to PFBC neurodegenerative disorder for which there are currently no targeting drugs and specific treatments available 5 , 23 – 25 . Lack of structural and related functional understanding limits patient mutation interpretation to disease mechanism. In this study, our structure and function analyses reveal that the pathogenic mutation residues (R459 and R570) line the phosphate translocation channel and act in phosphate recognition and transport. Notably, the MD simulations indicate that residue R570 interacts with Pi ion throughout the entire transport process (Fig. 3 d), underscoring the significance of R570. These mechanistic understanding might provide an opportunity for targeting XPR1 in the development of future therapeutics. XPR1 exhibits a channel-like architecture that facilitates phosphate efflux, distinguishing it from phosphate importers that use an alternating access mechanism for phosphate uptake 15 , 16 , 49 , 50 . This variety in mechanisms for balancing cellular phosphate levels offers cell regulatory layers. The activity of phosphate importers is regulated by the concentration gradient of other co-translocated ions (e.g., Na + or proton) 15 – 17 , 19 , but no co-translocated ions are required for XPR1 function 3 , 18 , 19 . Instead, XPR1 harbors an additional cytoplasmic SPX domain, which modulates XPR1 function by sensing PP-InsP nutrient messengers 1 , 18 , 30 – 32 . PP-InsPs have emerged as cellular high-Pi signals in regulating phosphate homeostasis 33 – 35 , 51 , 52 . The limited cryo-EM density observed for the SPX domain in our reconstructed map suggests its mobility. This mobile nature might facilitate PP-InsPs targeting and regulation. Further dynamic studies are needed to understand the regulatory role and mechanism of XPR1 SPX domain. Methods Protein expression and purification Human XPR1 DNA was subcloned into a pMlink vector encompassing a C-terminal 3×Flag tandem affinity tag. Point mutations were introduced into XPR1 genes by overlapping PCR and were verified by DNA sequencing. Proteins were expressed in Expi293F TM cells (Invitrogen) by transient transfection. Cells grown in Union-293 media (Union-Biotech, Shanghai) were transfected with linear polyethyleneimine (PEI) (Polysciences) at a cell density of 2.0×10 6 cells per ml -1 . The transfected cells were cultured for another 60 hours before harvesting. To preparation the Cryo-EM sample, cultured cells were collected and resuspended in the TBS buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM InsP 6 (a commercially available surrogate for the bioactive PP-InsPs 45 ), 1% LMNG (Anatrace), 0.1% CHS (Anatrace) and 0.25% Soy Phospholipids (Sigma). The extraction were performed at 4 °C for 1.5 h, and the resultant solution was centrifuged at 23,000 g for 40 min. The supernatant was collected and incubated with anti-Flag G1 affinity resin (Genscript) at 4 °C for 40 min, further rinsed with 30 bed volumes of wash (W1) buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM InsP 6 and 0.02% GDN (Anatrace), and eluted by W1 buffer supplemented with 250 μg ml -1 Flag peptide (Genscript). The eluent was concentrated and further purified by size-exclusion chromatography (Superose-6 Increase 10/300 column, GE Healthcare) using a buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM DTT, 1 mM InsP 6 and 0.02% GDN. The peak fractions of XPR1 were collected and pooled to ~4.5 mg ml −1 for Cryo-EM grid preparation. The Q576A mutant XPR1, with additional T580A/T582A substitutions for improving protein yield, was prepared in the same way as wild-type. Sample used for the liposome-based transport assays were extracted from cultured cells by a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl and 1% DDM. Target protein were further purified using anti-Flag G1 affinity resin and Superose-6 Increase 10/300 column in tandem, and prepared in the buffer containing 25 mM Hepes-Tris (pH 7.4), 150 mM NaCl, 2 mM DTT and 0.02% DDM for further proteoliposomes reconstitution. Cryo-EM grid preparation and data collection 3.5 μl aliquots of the purified protein was dropped onto glow discharged holey carbon grid (Quantifoil Cu R1.2/1.3, 300 mesh). The gird was blotted with a Vitrobot Mark IV (ThemoFisher Scientific) using 3.5 s blotting time with 100% humidity at 8 °C, and was plunge-frozen in liquid ethane. The cryo-grid was transferred to 300 kV Titan Krios electron microscopes (Thermo Fisher) equipped with a GIF Quantum energy filter (slit width 20 eV) and a Gatan K3 Summit detector. EPU software (v2.9) was used for fully automated data collection. Micrographs were recorded in the super-resolution mode with a magnification of 81,000×. Each micrograph stack, which contains 32 frames, was exposed for 3.5 s with a total electron dose of 50 e − /Å 2 . MotionCor2 (v1.4.7) 48 was used to perform beam-induced motion correction on cryo-EM images with binning factor of 2, resulting in a pixel size of 1.07 Å. The defocus value of each image was set to −1.2 to −2.2 μm and estimated by CTFFIND4 (v4.1.14) 49 . Cryo-EM data processing Diagrams of the procedures for data processing were described in Supplementary Fig. 2. For the structure determination of wild-type XPR1, 10,055 micrographs were manually selected from the original dataset of 10,255 micrographs. A total of 6,984,666 particles were selected and extracted for 2D classification, out of which 6,321,192 particles were selected for 3D classification. After several rounds of 3D classification, the particle with the best class was re-extracted to its original size for 3D refinement, resulting in a cryo-EM density map with an overall resolution of 3.5 Å. Further application of C2 symmetry yielded a 3.3 Å cryo-EM map, allowing for a clear visualization of the transmembrane domain of XPR1. For the structure determination of XPR1 mutant, we utilized 1,424,844 particles in a 3D classification using the "multi-reference" approach, which was derived from the wild-type XPR1. After 3D refinement, we reconstructed a cryo-EM map at a 3.2 Å resolution with C2 symmetry, sourced from 487,820 particles. CryoSPARC (v4.1) 50 and RELION (v4.0) 51 were used for 2D classification, 3D classification and 3D refinement. Local resolution variations of the maps were estimated using Resmap (v1.1.4) 52 . Model building and refinement The initial model of XPR1 was predicted from the Alphafold2 40 . We employed the ChimeraX software to dock this predicted model into the reconstructed cryo-EM map. The model was manually refined by iterative rounds of model adjusting in COOT 53 . The residues of XPR1 transmembrane domain (including 229-431 and 446-619) can be effectively constructed in the model. The obtained model was refined against the map using PHENIX 54 in real space with secondary and geometry restrains. Model quality assessments were conducted via Molprobity scores 55 and Ramachandran plots. Structural Figures were generated using ChimeraX (v1.6.1) and Pymol (v2.4.1). Transport assay Liposomes (10 mg/mL) were prepared with Ecoli total extract (Avanti Polar Lipids) in a reconstitution buffer containing 10 mM Hepes-Tris (pH 7.4) and 100 mM KCl. Preformed liposomes were dissolved with 1.3 % (w/v) DDM and mixed with purified XPR1 or variants in a protein-to-lipid ration of 1:100 (w/w). Following incubation at 4 °C for 1.5 h, the DDM was removed by 3 additions of SM-2 bio-beads (Bio-Rad), incubated for 2h/2h/overnight, respectively. Prior to the start of the transport assay, the proteoliposomes were extruded using polycarbonate filter with a pore size of 200 nm (Whatman). 15 μl proteoliposomes containing 0.2-0.5 μg protein were diluted into 80 μl reconstitution buffer. 32 Pi transport reactions were initiated by adding 50 μM KPi mixture, 10-25 μM [ 32 P] KH 2 PO 4 (3.7 MBq/ μmol; PerkinElmer) included. The assays were performed at room temperature for 6 min, and terminated by diluting tenfold with ice-cold stop buffer (10 mM Hepes-Tris, pH 7.4, 100 mM KCl and 5 mM non-labeled KH 2 PO 4 ), followed by rapid filtration through nitrocellulose membrane (Millipore, 0.22 μm Triton-free MCE). The filters were subsequently washed with 2×5 ml ice-cold stop buffer, placed in 5 mL Optiphase HiSafe 3 scintillation fluid and counted after 14 h. Background was defined as the counts of parallel transport assays that are terminated at the beginning of the reaction. After subtracting the background, the amount of phosphate transported inside the proteoliposomes was quantified with comparison to a standard curve for the substrate. The protein contained in proteoliposomes were resolved by SDS-PAGE and quantified using ImageJ. The Pi transport activity is determined by measuring phosphate uptake into proteoliposomes containing proteins (pmol Pi/μg protein). Each assay was performed a minimal of three times to generate an overall mean and s.d. Declarations Data availability The EM maps of wild-type and mutant XPR1 have been deposited in the Electron Microscopy Data Bank (www.ebi.ac.uk/pdbe/emdb/) with the accession numbers EMD-37205 and EMD-37239, respectively. The atomic coordinates of wild-type and mutant XPR1 structure models have been deposited in the Protein Data Bank (www.rcsb.org) with the accession codes 8KFM and 8KHB, respectively. Materials are available from the corresponding authors on request. Acknowledgments We thank the Cryo-EM Center, the University of Science and Technology of China (USTC), for the EM facility support. We are grateful to Dr. Yongxiang Gao (USTC) for technical support during EM image acquisition. We thank the Center for Protein Research, and Dr. Jianbo Cao at the Public Laboratory of Electron Microscopy, Huazhong Agricultural University, for technical support. We thank Prof. Michael Hothorn (University of Geneva) for critical comments on the manuscript. This work was supported by the National Natural Science Foundation of China (32071226 to Z.L.), the Foundation of Hubei Hongshan Laboratory (2021HSZD011 and 2021HSZD016 to Z.L. and P.Y.), and the HZAU-AGIS Cooperation Fund (SZYJY2022022 to Z.L.). Z.G. acknowledges the support of National Postdoctoral Program for Innovative Talents (BX2021108). Author contributions Z.L. conceived and supervised the project. W.Z., Y.C. and Z.G. designed all experiments. W.Z. prepared samples. Y.C. performed transport assays. Z.G. determined the structures Z.D., M.C., J.Z., J.Z. and P.C. contributed to plasmids constructing and data collecting. Q.W., Y.L., D.Z. and P.Y. contributed in data analysis. Z.L. and Y.C. wrote the manuscript with help from all authors. Competing interests The authors declare no competing interests. Correspondence and requests for materials should be addressed to Zhu Liu ( [email protected] ) References Giovannini D, Touhami J, Charnet P, Sitbon M, Battini JL (2013) Inorganic Phosphate Export by the Retrovirus Receptor XPR1 in Metazoans. Cell Rep 3:1866–1873 Hurlimann BC, Stadler-Waibel M, Werner TP, Freimoser FM (2007) Pho91 is a vacuolar phosphate phosphate and polyphosphate Saccharomyces cerevisiae. 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Nat Methods 16:670–673 Briones R, Biau C, Kutzner C, de Groot BL, Aponte-Santamaría C (2019) GROmaρs: A GROMACS-Based Toolset to Analyze Density Maps Derived from Molecular Dynamics Simulations. Biophys J 116:4–11 Additional Declarations There is NO Competing Interest. Supplementary Files SI20230821.pdf Supplementary Information SI20240816.docx Supplementary information SupplementaryVideo1.mp4 Supplementary video 1 Cite Share Download PDF Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3282549","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":341257168,"identity":"7a9e6f2f-c607-428f-a3fc-7630285f88e1","order_by":0,"name":"Zhu 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12:56:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3282549/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3282549/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-55471-9","type":"published","date":"2025-01-02T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63525159,"identity":"6f1efb86-4d69-4114-b8fc-8bd91ee9244b","added_by":"auto","created_at":"2024-08-29 06:53:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":487922,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructure of XPR1 transmembrane domain bound to phosphate.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003eCartoon representation of the structure. The N domain and EXS domain are colored in magenta and blue, respectively. TM9b is colored in yellow. The two phosphate ions are shown as sticks. \u003cstrong\u003eb\u003c/strong\u003eSchematic topology diagram of the structure. The gray background indicates the membrane bilayer.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3282549/v1/bb82a7f5ff5babba3576fbd3.png"},{"id":63525162,"identity":"9a9b18d6-be6d-43a7-b9d0-543e3cdc4f3e","added_by":"auto","created_at":"2024-08-29 06:53:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":557688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhosphate recognition and transport by XPR1.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Channel-like architecture. Only the channel-forming transmembrane α-helices (TM2, TM5 and TM6-TM10) are represented for clarity. The solvent-accessible pathway (gray dots representation) is calculated using HOLE\u003ca href=\"#_ENREF_33\" title=\"Smart, 1996 #40\"\u003e\u003csup\u003e33\u003c/sup\u003e\u003c/a\u003e, and the calculated channel radii are shown on the right. The gray dashed line indicates a radius of 1 Å for water access limit. \u003cstrong\u003eb\u003c/strong\u003e A cut-open electrostatic surface representation of the channel. It is colored in terms of electrostatic potential, and displayed in a scale from red (−5 kT/e) to blue (+5 kT/e). Phosphate ions are show as green sticks.\u003cstrong\u003e c\u003c/strong\u003e The two binding sites of phosphate ions. The central axis of TM9a is indicated by a gray stick, for highlighting the kink of TM9 at W573. \u003cstrong\u003ed\u003c/strong\u003e Activity assay. The Pi transport activity is determined by measuring phosphate uptake into liposomes containing wild-type and variant forms of XPR1. Activity of PFBC families variants, R570C and R459C, are highlighted in red. The data presented are the average of three independent measurements, and the error indicates SD.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3282549/v1/0535231ad79229e7113179ac.png"},{"id":63525164,"identity":"c520b08d-6d12-4eee-91ab-e29b453e50b9","added_by":"auto","created_at":"2024-08-29 06:53:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":273137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMovement of phosphate ion within the translocation channel. a\u003c/strong\u003e Local conformational changes. The structures of wild-type and mutant XPR1 are superposed, with wild-type colored in the same scheme as Fig. 2c and mutant form in light green, respectively. Other portions of the two structures are omitted for clarity. The EM densities of Pi\u003csub\u003e1\u003c/sub\u003e and W573 in the two structures are indicated on the right, contoured at 5.5 s. \u003cstrong\u003eb\u003c/strong\u003e Activity assay. The Pi transport activity of wild-type and variant forms of XPR1 are averaged from three independent measurements, and the error indicates SD. \u003cstrong\u003ec\u003c/strong\u003e Cartoon representation of the XPR1 \"ring flipping-push-movement\" mechanism for propelling Pi ions trough the translocation channel.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3282549/v1/b5fb1a746bd7c81b1c324ae6.png"},{"id":72949905,"identity":"7c2b083e-52ed-455c-a869-d628795a9529","added_by":"auto","created_at":"2025-01-04 08:13:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1821511,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3282549/v1/6f98aa52-7a18-410c-b1ac-83bbaf5ade2f.pdf"},{"id":63525674,"identity":"ecdc86d0-3084-4e2a-82b8-4336b6bfa5e5","added_by":"auto","created_at":"2024-08-29 07:01:12","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2080480,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SI20230821.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3282549/v1/937957f20041c10e9cdedd88.pdf"},{"id":63525166,"identity":"1e8105f1-a70f-4f97-986d-735d15438d1e","added_by":"auto","created_at":"2024-08-29 06:53:12","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11484998,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"SI20240816.docx","url":"https://assets-eu.researchsquare.com/files/rs-3282549/v1/db229e4515ea327861ba6927.docx"},{"id":63525168,"identity":"3742f27e-e2c7-4b3c-9a31-1d2855c36be5","added_by":"auto","created_at":"2024-08-29 06:53:13","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3581383,"visible":true,"origin":"","legend":"Supplementary video 1","description":"","filename":"SupplementaryVideo1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3282549/v1/d275c4b37a25d12aed48b6ed.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Structural insights into the mechanism of phosphate recognition and transport by human XPR1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInorganic phosphate (Pi) is essential for life, used wildly in biomolecules synthesis, cell metabolism and energy supply. To nourish cell, humans uptake Pi by the solute carrier 20 (SLC20) and SLC34 transporter families\u003csup\u003e6-10\u003c/sup\u003e. In parallel, cell exports intracellular Pi to regulate Pi homeostasis and prevent the cytotoxicity caused by Pi accumulation. Elevated Pi levels cause major disorders, with severe biochemical and clinical consequences\u003csup\u003e11-13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eXPR1 is the only known exporter that transports Pi out of cell in humans\u003csup\u003e1\u003c/sup\u003e, expressed in all tissues\u003csup\u003e14\u003c/sup\u003e. Loss-of-function variants of XPR1 accumulate cytosolic Pi and lead to cerebral calcium-phosphate deposition, which are associated with a genetic disease known as primary familial brain calcification (PFBC)\u003csup\u003e5\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e15-17\u003c/sup\u003e. PFBC is an adult-onset neurodegenerative disorder characterized by a large clinically heterogeneous symptoms including dystonia, parkinsonism, dementia, depression, chorea and others, yet no targeting drugs and specific treatment are currently available\u003csup\u003e18-20\u003c/sup\u003e. XPR1 is abundant in human platelets, where inhibiting its transport activity increases the risk of thrombosis in mouse models\u003csup\u003e21\u003c/sup\u003e. XPR1 deficiency in ovarian cancer cell causes toxic accumulation of cytosolic Pi, leading to cell death\u003csup\u003e22\u003c/sup\u003e. Dysregulation of Pi efflux could thus presents a new, potential strategy for anticancer therapy\u003csup\u003e22\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e23\u003c/sup\u003e. Given the significance and pathomechanisms of XPR1, it presents a promising therapeutic target for diseases and cancers\u003csup\u003e5\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e22\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e24\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e25\u003c/sup\u003e. However, the molecular basis by which XPR1 recognizes and transports Pi across cell membrane remains unknown. We therefore set out to determine the Cryo-EM structure of human XPR1 bound to Pi.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe overall Cryo-EM structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXPR1 contains a transmembrane domain and a cytoplasmic N-terminal SPX domain. The transmembrane domain is responsible for facilitating Pi efflux, while the SPX domain enhances this function by binding inositol pyrophosphate (PP-InsP) stimuli\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e26-29\u003c/sup\u003e. By preparing XPR1 in the presence of PP-InsP (Supplementary Fig. 1, and Methods), we could collect high-quality cryo-EM images for single particle analysis, which allowed us to reconstruct a density map with an overall resolution of 3.3 \u0026Aring; (Supplementary Fig. 2a). The EM density of SPX domain is poor (Supplementary Fig. 3a), probably owing to its mobility. In contrast, the density map of transmembrane domain is clear (Supplementary Fig. 3a). Although the transmembrane domain is small, with a molecule weight of only approximately 43 kDa, the well resolved EM density enables us to build the atomic model (Supplementary Fig. 3b, c, and Supplementary Table 1).\u003c/p\u003e\n\u003cp\u003eThe transmembrane domain of XPR1 contains 10 transmembrane \u0026alpha;-helices (TM1-10) and folds into two structurally distinct sub-domains, with N and C termini on the intracellular side (Fig. 1). We refer to the N-terminal portion as N domain, that is formed by TM1-TM5 and a short amphipathic helix (AH) lying parallel to the membrane. The C-terminal portion harbors the conserved EXS (named for homologous regions found in yeast ERD1 and SYG1 and human XPR1) domain\u003csup\u003e30\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e31\u003c/sup\u003e (Supplementary Fig. 4), that is made up of TM6-TM10. The EXS domain associates with TM5, creating a pore that spans across the membrane (Fig. 1a). Lined the membrane-spanning pore, two phosphate ions were identified (Fig. 1a, Supplementary Fig. 3d), implying that the pore serves to transport Pi. Notably, TM9b positions close to the central pore axis on the extracellular side (Fig. 1a), suggesting its potential role in Pi exporting. A 3-dimensional structural homology search with the program DALI\u003csup\u003e32\u003c/sup\u003e found no known structures similar to the XPR1 transmembrane domain, indicating a specific mechanism for phosphate recognition and transport in XPR1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe structure basis for phosphate recognition and transport\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHOLE\u003csup\u003e33\u003c/sup\u003e analysis for the solvent-accessible pathway of XPR1 transmembrane domain reveals that it exhibits an ion channel-like architecture for Pi transit (Fig. 2a, b). The intracellular entrance of the translocation channel is formed by TM5a, TM6-TM8 and TM10 (Fig. 2a). Owing to a kink in TM9, the channel is bent and created by TM9b, TM10, TM6, TM5b and the tip of TM2 on the extracellular side. The narrowest point of the channel has a radius of approximately 1.2 \u0026Aring;, which exceeds the water access limit (1 \u0026Aring;)\u003csup\u003e34\u003c/sup\u003e, indicating that the channel is solvent-permeable. The entrance and the interior wall of the first half of the channel are positively charged, while the second half and the exit are negatively charged (Fig. 2b). Given the anionic property of Pi ions, the presence of polarized electrostatic potential across the translocation channel may serve to move Pi ions in and out. The solvent-accessibility of the translocation channel (Fig. 2a), together with aligned Pi ions along the permeation pathway (Fig. 2b), suggests that XPR1 may export Pi ions through this channel without requiring large conformational changes as observed in Pi importers. Pi importers exhibit an outward-open conformation for Pi uptake from extracellular side and undergo large conformational changes to adopt an inward-open conformation, allowing for Pi release into cytosol\u003csup\u003e35-37\u003c/sup\u003e. This conformational transition is driven by the energy derived from movements of other co-translocated ions (e.g., Na\u003csup\u003e+\u003c/sup\u003e or protons) down their concentration gradients\u003csup\u003e35-37\u003c/sup\u003e. In contrast, the Pi export activity of XPR1 is independent of pH gradient across the cell membrane\u003csup\u003e3\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e27\u003c/sup\u003e, and no co-translocated ions have been found\u003csup\u003e23\u003c/sup\u003e. These previously observed phenomena are now supported and aligned with by the channel-like architecture of XPR1.\u003c/p\u003e\n\u003cp\u003eOur structure provides a framework for understanding the mechanism by which XPR1 recognizes phosphate. Two phosphate ions are recognized by a series of polar residues in the translocation channel (Fig. 2c). One of them (Pi\u003csub\u003e1\u003c/sub\u003e) interacts with the side chains of N401, R570, Q576 and E600, and is coordinated downstream (intracellular-to-extracellular direction) of the other Pi ion (Pi\u003csub\u003e2\u003c/sub\u003e). The Pi\u003csub\u003e2\u003c/sub\u003e is recognized by interacting with D398, K482, Y483, D533, R570, R603, R604 and W607. Additionally, the side chain of R459 positions the orientation of D398 and Y483, facilitating them to coordinate Pi\u003csub\u003e2\u003c/sub\u003e in the binding site. Moreover, the side chain of R570 points towards the midpoint between the two Pi ions, bridging them, which implies its potential role in Pi delivery during translocation. There is a discontinuity in TM9 that is kinked at W573. The TM9b is stabilized by an intrahelical hydrogen bond between W573 and Q576, as well as the interaction between Q576 and Pi\u003csub\u003e1\u003c/sub\u003e. These Pi-recognizing residues are conserved in different species (Supplementary Fig. 4).\u003c/p\u003e\n\u003cp\u003eTo assess the roles of the above-mentioned key residues in Pi transport, we reconstituted XPR1 into liposomes and performed Pi transport assays (Methods). By substituted the Pi\u003csub\u003e2\u003c/sub\u003e-recognizing residues D398, K482, D533, R603 and R604 with alanine, and Y483 and W607 with phenylalanine, and could purify the R603A or the Y483F in sufficient quantities to perform the proteoliposome-based transport assays. We find that XPR1 carrying either R603A or Y483F substitution have impaired Pi transport activities (Fig. 2d), suggesting crucial roles of Y483 and R603 in transporting Pi. Moreover, consistent with the structure configuration of the Pi\u003csub\u003e1\u003c/sub\u003ebinding site (Fig. 2c), replacing side chain of N401, Q576 or E600 with alanine resulted in a reduced Pi transport activity of XPR1 (Fig. 2d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinically linked residues in XPR1 function in phosphate recognition and transport\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eXPR1\u003c/em\u003e is a causative gene identified in primary familial brain calcification (PFBC) families\u003csup\u003e5\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e16\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e17\u003c/sup\u003e, and the R459C and R570C missense variants have been found to be pathogenic\u003csup\u003e15\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e20\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e38\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e39\u003c/sup\u003e. Our structure reveals that R459 is involved in maintaining the binding network of Pi\u003csub\u003e2\u003c/sub\u003e, and R570 forms salt bridges with Pi\u003csub\u003e2\u003c/sub\u003e and Pi\u003csub\u003e1\u003c/sub\u003e (Fig. 2c). Substituting the side chains of R459 and R570 might perturb Pi binding and potentially impair XPR1 activity. Indeed, we find that the PFBC families variants, R570C and R459C, exhibit an significant reduction in Pi transport activity (Fig. 2d). Therefore, our structure and function analysis provide mechanistic insights into the correlation between patient mutations and XPR1 function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe mechanism for linking phosphate recognition and transport\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHow do the phosphate ions pass through the XPR1 transmembrane channel? A structure comparison between channel alone and bound to phosphate ions would provide mechanistic insights. With the resolved experimental structure of the phosphate-bound channel at hand, we compared it with the unbound state model predicted by AlphaFold\u003csup\u003e40\u003c/sup\u003e. While the AlphaFold model and our experimental structure share the overall fold, a notable disparity is observed in the TM9 segment (Supplementary Fig. 5). The experimental structure has a kink in this segment, causing a shift of TM9b towards the central membrane-spanning pore axis, while the AlphaFold model predicts an unbent TM9. We reasoned that the TM9 kink in the experimental structure is stabilized by the intrahelical hydrogen bond formed between the side chains of Q576 and W573, and the interaction between Q576 and the bound phosphate (Pi\u003csub\u003e1\u003c/sub\u003e) (Fig. 2c). We therefore speculated that substituting Q576 side chain with alanine would disrupt the intrahelical hydrogen bond and lead to the Pi\u003csub\u003e1\u003c/sub\u003e dissociation. To clarify these envisioned conformational changes, we sought to determine the Cryo-EM structure of the Q576A mutant XPR1.\u003c/p\u003e\n\u003cp\u003eThe transmembrane domain structure of the XPR1 Q576A mutant was resolved at a resolution of 3.2 \u0026Aring; (Supplementary Fig. 2b, and Methods). This mutant presents a similar overall structure to that of wild-type, and also two phosphate ions are observed in the translocation channel (Supplementary Fig. 6). Notably, local conformational changes in the Pi\u003csub\u003e1\u003c/sub\u003e binding site are revealed (Fig. 3a). In the mutant structure, the Pi\u003csub\u003e1\u003c/sub\u003e ion undergoes a forward movement (intracellular-to-extracellular direction) within the translocation channel. Consequently, the hydrogen bonds between Pi\u003csub\u003e1\u003c/sub\u003e and the side chains of N401 and E600 are broken. Moreover, the indole ring of W573 flips to form an intrahelical hydrogen bond with R570. This rearranged intrahelical hydrogen bond disrupts the salt bridge between R570 and Pi\u003csub\u003e1\u003c/sub\u003e, and thus the Pi\u003csub\u003e1 \u003c/sub\u003edissociation and forward movement. Additionally, given the high density of \u0026pi;-electrons in the W573 indole ring, the ring-flipping and closing to the Pi\u003csub\u003e1\u003c/sub\u003e ion would push the anion forward. The ring-flipping therefore links Pi recognition to transport. Supporting this, when the indole ring of W573 is substituted with polar or hydrophobic group like W573N, W573Y, W573A and W573L, the Pi transport activity of XPR1 is reduced (Fig. 3b). Together, the translocation channel likely employ a \u0026quot;ring flipping-push-movement\u0026quot; mechanism to propel Pi ions through, facilitated by the rearrangement of two intrahelical hydrogen bonds between W573-Q576 and W573-R570 (Fig. 3c). As Q576 is replaced by alanine in the mutant structure, a possibility cannot be ruled out that the side chain of Q576 also plays role in the Pi forward movement in native protein.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eElevated levels of cytosolic phosphate are cytotoxic due to its presence as a potent metal chelator and a pervasive inhibitor of cellular enzymes. XPR1 and its evolutionarily conserved orthologues are the proteins that transport cytosolic phosphate out of cells\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. A very recent study discovered that the XPR1 orthologue in fruit fly (Pxo) lowers cellular phosphate levels, by generating a newly defined phosphate-storing organelle (PXo body) and transporting cytosolic phosphate into the PXo body\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. These transporters belong to the structurally uncharacterized SLC53 family. Here the experimental structure of XPR1 transmembrane domain, together with its multiple functional states bound to phosphate, provides the first snapshots for understanding the structure-function relationships of SLC53 family transporters.\u003c/p\u003e \u003cp\u003eXPR1 mutations are linked to PFBC neurodegenerative disorder for which there are currently no targeting drugs and specific treatments available\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Lack of structural and related functional understanding limits patient mutation interpretation to disease mechanism. In this study, our structure and function analyses reveal that the pathogenic mutation residues (R459 and R570) line the phosphate translocation channel and act in phosphate recognition and transport. Notably, the MD simulations indicate that residue R570 interacts with Pi ion throughout the entire transport process (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), underscoring the significance of R570. These mechanistic understanding might provide an opportunity for targeting XPR1 in the development of future therapeutics.\u003c/p\u003e \u003cp\u003eXPR1 exhibits a channel-like architecture that facilitates phosphate efflux, distinguishing it from phosphate importers that use an alternating access mechanism for phosphate uptake\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. This variety in mechanisms for balancing cellular phosphate levels offers cell regulatory layers. The activity of phosphate importers is regulated by the concentration gradient of other co-translocated ions (e.g., Na\u003csup\u003e+\u003c/sup\u003e or proton)\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, but no co-translocated ions are required for XPR1 function\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Instead, XPR1 harbors an additional cytoplasmic SPX domain, which modulates XPR1 function by sensing PP-InsP nutrient messengers\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. PP-InsPs have emerged as cellular high-Pi signals in regulating phosphate homeostasis\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The limited cryo-EM density observed for the SPX domain in our reconstructed map suggests its mobility. This mobile nature might facilitate PP-InsPs targeting and regulation. Further dynamic studies are needed to understand the regulatory role and mechanism of XPR1 SPX domain.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eProtein expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman XPR1 DNA was subcloned into a pMlink vector encompassing a C-terminal 3×Flag tandem affinity tag. Point mutations were introduced into XPR1 genes by overlapping PCR and were verified by DNA sequencing. Proteins were expressed in Expi293F\u003csup\u003eTM\u003c/sup\u003e cells (Invitrogen) by transient transfection. Cells grown in Union-293 media (Union-Biotech, Shanghai) were transfected with linear polyethyleneimine (PEI) (Polysciences) at a cell density of 2.0×10\u003csup\u003e6\u003c/sup\u003e cells per ml\u003csup\u003e-1\u003c/sup\u003e. The transfected cells were cultured for another 60 hours before harvesting.\u003c/p\u003e\n\u003cp\u003eTo preparation the Cryo-EM sample, cultured cells were collected and resuspended in the TBS buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM InsP\u003csub\u003e6\u003c/sub\u003e (a commercially available surrogate for the bioactive PP-InsPs\u003ca href=\"#_ENREF_45\" title=\"Guan, 2023 #44\"\u003e\n \u003csup\u003e45\u003c/sup\u003e\n \u003c/a\u003e), 1% LMNG (Anatrace), 0.1% CHS (Anatrace) and 0.25% Soy Phospholipids (Sigma). The extraction were performed at 4 °C for 1.5 h, and the resultant solution was centrifuged at 23,000 g for 40 min. The supernatant was collected and incubated with anti-Flag G1 affinity resin (Genscript) at 4 °C for 40 min, further rinsed with 30 bed volumes of wash (W1) buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM InsP\u003csub\u003e6\u003c/sub\u003e and 0.02% GDN (Anatrace), and eluted by W1 buffer supplemented with 250 μg ml\u003csup\u003e-1\u003c/sup\u003e Flag peptide (Genscript). The eluent was concentrated and further purified by size-exclusion chromatography (Superose-6 Increase 10/300 column, GE Healthcare) using a buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM DTT, 1 mM InsP\u003csub\u003e6\u003c/sub\u003e and 0.02% GDN. The peak fractions of XPR1 were collected and pooled to ~4.5 mg ml\u003csup\u003e−1\u003c/sup\u003e for Cryo-EM grid preparation. The Q576A mutant XPR1, with additional T580A/T582A substitutions for improving protein yield, was prepared in the same way as wild-type.\u003c/p\u003e\n\u003cp\u003eSample used for the liposome-based transport assays were extracted from cultured cells by a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl and 1% DDM. Target protein were further purified using anti-Flag G1 affinity resin and Superose-6 Increase 10/300 column in tandem, and prepared in the buffer containing 25 mM Hepes-Tris (pH 7.4), 150 mM NaCl, 2 mM DTT and 0.02% DDM for further proteoliposomes reconstitution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM grid preparation and data collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3.5 μl aliquots of the purified protein was dropped onto glow discharged holey carbon grid (Quantifoil Cu R1.2/1.3, 300 mesh). The gird was blotted with a Vitrobot Mark IV (ThemoFisher Scientific) using 3.5 s blotting time with 100% humidity at 8 °C, and was plunge-frozen in liquid ethane. The cryo-grid was transferred to 300 kV Titan Krios electron microscopes (Thermo Fisher) equipped with a GIF Quantum energy filter (slit width 20 eV) and a Gatan K3 Summit detector. EPU software (v2.9) was used for fully automated data collection. Micrographs were recorded in the super-resolution mode with a magnification of 81,000×. Each micrograph stack, which contains 32 frames, was exposed for 3.5 s with a total electron dose of 50 e\u003csup\u003e−\u003c/sup\u003e/Å\u003csup\u003e2\u003c/sup\u003e. MotionCor2 (v1.4.7)\u003ca href=\"#_ENREF_48\" title=\"Zheng, 2017 #57\"\u003e\n \u003csup\u003e48\u003c/sup\u003e\n \u003c/a\u003e was used to perform beam-induced motion correction on cryo-EM images with binning factor of 2, resulting in a pixel size of 1.07 Å. The defocus value of each image was set to −1.2 to −2.2 μm and estimated by CTFFIND4 (v4.1.14)\u003ca href=\"#_ENREF_49\" title=\"Rohou, 2015 #59\"\u003e\n \u003csup\u003e49\u003c/sup\u003e\n \u003c/a\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM data processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiagrams of the procedures for data processing were described in Supplementary Fig. 2. For the structure determination of wild-type XPR1, 10,055 micrographs were manually selected from the original dataset of 10,255 micrographs. A total of 6,984,666 particles were selected and extracted for 2D classification, out of which 6,321,192 particles were selected for 3D classification. After several rounds of 3D classification, the particle with the best class was re-extracted to its original size for 3D refinement, resulting in a cryo-EM density map with an overall resolution of 3.5 Å. Further application of C2 symmetry yielded a 3.3 Å cryo-EM map, allowing for a clear visualization of the transmembrane domain of XPR1. For the structure determination of XPR1 mutant, we utilized 1,424,844 particles in a 3D classification using the \"multi-reference\" approach, which was derived from the wild-type XPR1. After 3D refinement, we reconstructed a cryo-EM map at a 3.2 Å resolution with C2 symmetry, sourced from 487,820 particles. CryoSPARC (v4.1)\u003ca href=\"#_ENREF_50\" title=\"Punjani, 2017 #60\"\u003e\n \u003csup\u003e50\u003c/sup\u003e\n \u003c/a\u003e and RELION (v4.0)\u003ca href=\"#_ENREF_51\" title=\"Kimanius, 2021 #61\"\u003e\n \u003csup\u003e51\u003c/sup\u003e\n \u003c/a\u003e were used for 2D classification, 3D classification and 3D refinement. Local resolution variations of the maps were estimated using Resmap (v1.1.4)\u003ca href=\"#_ENREF_52\" title=\"Kucukelbir, 2014 #62\"\u003e\n \u003csup\u003e52\u003c/sup\u003e\n \u003c/a\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModel building and refinement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe initial model of XPR1 was predicted from the Alphafold2\u003ca href=\"#_ENREF_40\" title=\"Jumper, 2021 #43\"\u003e\n \u003csup\u003e40\u003c/sup\u003e\n \u003c/a\u003e. We employed the ChimeraX software to dock this predicted model into the reconstructed cryo-EM map. The model was manually refined by iterative rounds of model adjusting in COOT\u003ca href=\"#_ENREF_53\" title=\"Brown, 2015 #63\"\u003e\n \u003csup\u003e53\u003c/sup\u003e\n \u003c/a\u003e. The residues of XPR1 transmembrane domain (including 229-431 and 446-619) can be effectively constructed in the model. The obtained model was refined against the map using PHENIX\u003ca href=\"#_ENREF_54\" title=\"Afonine, 2018 #64\"\u003e\n \u003csup\u003e54\u003c/sup\u003e\n \u003c/a\u003e in real space with secondary and geometry restrains. Model quality assessments were conducted via Molprobity scores\u003ca href=\"#_ENREF_55\" title=\"Williams, 2018 #65\"\u003e\n \u003csup\u003e55\u003c/sup\u003e\n \u003c/a\u003e and Ramachandran plots. Structural Figures were generated using ChimeraX (v1.6.1) and Pymol (v2.4.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransport assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiposomes (10 mg/mL) were prepared with Ecoli total extract (Avanti Polar Lipids) in a reconstitution buffer containing 10 mM Hepes-Tris (pH 7.4) and 100 mM KCl. Preformed liposomes were dissolved with 1.3 % (w/v) DDM and mixed with purified XPR1 or variants in a protein-to-lipid ration of 1:100 (w/w). Following incubation at 4 °C for 1.5 h, the DDM was removed by 3 additions of SM-2 bio-beads (Bio-Rad), incubated for 2h/2h/overnight, respectively. Prior to the start of the transport assay, the proteoliposomes were extruded using polycarbonate filter with a pore size of 200 nm (Whatman). 15 μl proteoliposomes containing 0.2-0.5 μg protein were diluted into 80 μl reconstitution buffer. \u003csup\u003e32\u003c/sup\u003ePi transport reactions were initiated by adding 50 μM KPi mixture, 10-25 μM [\u003csup\u003e32\u003c/sup\u003eP] KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (3.7 MBq/ μmol; PerkinElmer) included. The assays were performed at room temperature for 6 min, and terminated by diluting tenfold with ice-cold stop buffer (10 mM Hepes-Tris, pH 7.4, 100 mM KCl and 5 mM non-labeled KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), followed by rapid filtration through nitrocellulose membrane (Millipore, 0.22 μm Triton-free MCE). The filters were subsequently washed with 2×5 ml ice-cold stop buffer, placed in 5 mL Optiphase HiSafe 3 scintillation fluid and counted after 14 h. Background was defined as the counts of parallel transport assays that are terminated at the beginning of the reaction. After subtracting the background, the amount of phosphate transported inside the proteoliposomes was quantified with comparison to a standard curve for the substrate. The protein contained in proteoliposomes were resolved by SDS-PAGE and quantified using ImageJ. The Pi transport activity is determined by measuring phosphate uptake into proteoliposomes containing proteins (pmol Pi/μg protein). Each assay was performed a minimal of three times to generate an overall mean and s.d.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe EM maps of wild-type and mutant XPR1 have been deposited in the Electron Microscopy Data Bank (www.ebi.ac.uk/pdbe/emdb/) with the accession numbers EMD-37205 and EMD-37239, respectively. The atomic coordinates of wild-type and mutant XPR1 structure models have been deposited in the Protein Data Bank (www.rcsb.org) with the accession codes 8KFM and 8KHB, respectively. Materials are available from the corresponding authors on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Cryo-EM Center, the University of Science and Technology of China (USTC), for the EM facility support. We are grateful to Dr. Yongxiang Gao (USTC) for technical support during EM image acquisition. We thank the Center for Protein Research, and Dr. Jianbo Cao at the Public Laboratory of Electron Microscopy, Huazhong Agricultural University, for technical support. We thank Prof. Michael Hothorn (University of Geneva) for critical comments on the manuscript. This work was supported by the National Natural Science Foundation of China (32071226 to Z.L.), the Foundation of Hubei Hongshan Laboratory (2021HSZD011 and 2021HSZD016 to Z.L. and P.Y.), and the HZAU-AGIS Cooperation Fund (SZYJY2022022 to Z.L.). Z.G. acknowledges the support of National Postdoctoral Program for Innovative Talents (BX2021108).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.L. conceived and supervised the project. W.Z., Y.C. and Z.G. designed all experiments. W.Z. prepared samples. Y.C. performed transport assays. Z.G. determined the structures Z.D., M.C., J.Z., J.Z. and P.C. contributed to plasmids constructing and data collecting. Q.W., Y.L., D.Z. and P.Y. contributed in data analysis. Z.L. and Y.C. wrote the manuscript with help from all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence and requests for materials\u003c/strong\u003e should be addressed to Zhu Liu ([email protected])\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGiovannini D, Touhami J, Charnet P, Sitbon M, Battini JL (2013) Inorganic Phosphate Export by the Retrovirus Receptor XPR1 in Metazoans. Cell Rep 3:1866\u0026ndash;1873\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHurlimann BC, Stadler-Waibel M, Werner TP, Freimoser FM (2007) Pho91 is a vacuolar phosphate phosphate and polyphosphate Saccharomyces cerevisiae. 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J Chem Theory Comput 7:525\u0026ndash;537\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026aacute;ll S, Abraham MJ, Kutzner C, Hess B, Lindahl E (2015) Tackling exascale software challenges in molecular dynamics simulations with GROMACS. \u003cem\u003e2nd International Conference on Exascale Applications and Software (EASC).\u003c/em\u003e 3\u0026ndash;27\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonomi M et al (2019) Promoting transparency and reproducibility in enhanced molecular simulations. Nat Methods 16:670\u0026ndash;673\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBriones R, Biau C, Kutzner C, de Groot BL, Aponte-Santamar\u0026iacute;a C (2019) GROmaρs: A GROMACS-Based Toolset to Analyze Density Maps Derived from Molecular Dynamics Simulations. Biophys J 116:4\u0026ndash;11\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3282549/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3282549/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eXPR1 is the only known protein that transports inorganic phosphate (Pi) out of cells, and the function is conserved across species from yeast to mammals\u003ca href=\"#_ENREF_1\" title=\"Giovannini, 2013 #9\"\u003e\u003csup\u003e1-4\u003c/sup\u003e\u003c/a\u003e. Human XPR1 variants lead to cerebral calcium-phosphate deposition, which are associated with a neurodegenerative disorder known as primary familial brain calcification (PFBC)\u003ca href=\"#_ENREF_5\" title=\"Legati, 2015 #12\"\u003e\u003csup\u003e5\u003c/sup\u003e\u003c/a\u003e. Here, we present the Cryo-EM structure of human XPR1 bound to Pi ions. XPR1 contains 10 transmembrane α-helices, forming an ion channel-like architecture that recognizes and transports Pi ions. Two arginine residues, subject to pathogenic mutation in PFBC families, line the translocation channel and serve to bind Pi ions. Clinically linked mutations of these arginine residues impair the Pi transport activity of XPR1. To track the movement of Pi ions within the translocation channel, we capture a mutant XPR1 in an alternative conformation. It reveals a rearrangement of intrahelical hydrogen bonds between a channel-lining tryptophan and two Pi-binding residues. This rearrangement links Pi recognition and transport, by means of flipping the tryptophan residue to propel Pi through the translocation channel. Our results provide mechanistic understanding of how XPR1 recognizes and transports phosphate ions across cell membrane, and they establish a framework for interpreting disease-related mutations and for the development of future therapeutics.\u003c/p\u003e","manuscriptTitle":"Structural insights into the mechanism of phosphate recognition and transport by human XPR1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-29 06:53:07","doi":"10.21203/rs.3.rs-3282549/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2f9b3bfa-7da8-457f-96df-c57e10711b75","owner":[],"postedDate":"August 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":36161895,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":36161896,"name":"Biological sciences/Biochemistry/Proteins/Membrane proteins"}],"tags":[],"updatedAt":"2025-01-04T08:13:05+00:00","versionOfRecord":{"articleIdentity":"rs-3282549","link":"https://doi.org/10.1038/s41467-024-55471-9","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-01-02 05:00:00","publishedOnDateReadable":"January 2nd, 2025"},"versionCreatedAt":"2024-08-29 06:53:07","video":"","vorDoi":"10.1038/s41467-024-55471-9","vorDoiUrl":"https://doi.org/10.1038/s41467-024-55471-9","workflowStages":[]},"version":"v1","identity":"rs-3282549","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3282549","identity":"rs-3282549","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}